JP3379774B2 - Base sequence determination method - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for determining a base sequence of a nucleic acid, and more particularly to a method and a kit for determining a base sequence using a fluorescently labeled oligonucleotide as a primer.

[0002]

2. Description of the Related Art The dideoxy chain terminator method (dideoxy method), which has been widely used as a DNA base sequence analysis method, has conventionally been a method in which DNA is labeled with a radioactive element and a pattern obtained by gel electrophoresis is separated according to the base length by autoradio. Although it has been performed by reading with a graph, an automatic sequencer using a fluorescent substance label has been developed in recent years due to the development of peripheral technology, and various improvements have been made to obtain highly accurate data. In this method, a fluorescent substance is bound to an oligonucleotide used as a primer or dideoxynucleotide triphosphate as a reaction-terminating substrate, and the DNA fragment is irradiated with a laser beam while separating the dideoxy reaction product by gel electrophoresis, and emitted. It receives fluorescence and decodes it one base at a time. In general, the former primer labeling method is mainly used because it provides more accurate results and is easy to synthesize.

[0003]

In this primer labeling method, one molecule of the fluorescent substance is usually bound to one molecule of the primer and used. When using the fluorescent labeling method, the accuracy of the data depends especially on the signal intensity. In particular, the stronger the signal strength, the more advantageous it is to decode the data for a longer time in one reaction. To achieve this purpose, it is possible to introduce a plurality of fluorescent substances per molecule of primer. However, it is expected that the binding between the primer and the template DNA becomes unstable due to conformational change or steric hindrance, etc., and the fluorescence intensity decreases due to mutual interference of the fluorescent substances. The signal intensity does not always increase even if an arbitrary number of fluorescent substances are introduced at the position. That is, it is estimated that the introduction position and the number of the fluorescent substance are important factors. However, the optimum values of those factors are not known. An object of the present invention is to provide a fluorescent-labeled primer having a high signal intensity, to provide a highly sensitive and highly accurate nucleic acid nucleotide sequence determination method using the primer, and a kit used for the method.

[0004]

The present invention will be summarized. The first invention of the present invention relates to a method for determining a nucleotide sequence of a nucleic acid by the dideoxy method, at the 5'end and 4 to 12 from the 5'end.
It is characterized in that an oligonucleotide in which one molecule of a fluorescent substance is bound between nucleotides at positions separated by individual bases is used as a primer. The second aspect of the present invention
Invention was used to determine the base sequence of nucleic acids by the dideoxy method.
A primer used for the above oligonucleoside
Regarding tide primer. The third invention of the present invention relates to a kit for determining a nucleotide sequence of a nucleic acid, which is the above-mentioned second invention.
The oligonucleotide primer of the above is included.

The present invention will be described in detail below. The length of the oligonucleotide used as the primer of the present invention is
As long as it can be used in the dideoxy method,
Although not particularly limited, it is preferably 10 to 30 bases.

The base sequence of the oligonucleotide used as the primer of the present invention may be any of widely used vector-derived sequences, uniquely designed sequences and the like that can be used in the dideoxy method. . Examples of vector-derived sequences include M1, M2 and M represented by SEQ ID NOS: 1 to 10 in the sequence listing.
3, M4, RV, RVM, RVN, T3, T7, SP6
Each of the primers can be mentioned.

As a method for synthesizing an oligonucleotide,
Examples include the phosphorous amidite method, the phosphoric acid triester method, the hydrogenphosphonate method, and the like, but the phosphorous amidite method is common.

As a method for introducing a fluorescent substance into an oligonucleotide, a method in which an oligonucleotide having a linker arm bound thereto is synthesized and a labeling reagent is allowed to act on the oligonucleotide is generally used. Known reagents for the linker arm can be used, and some of them are commercially available. By appropriately selecting these linker arm introduction reagents, a linker arm can be introduced at the 5'end, between nucleotides, 3'end, base, etc. of an oligonucleotide to bind a fluorescent substance. For example, there is a compound represented by the following formula (Formula 1) as a reagent for introducing a linker arm at the 5 ′ end, and a compound represented by the following formula (Formula 2) as a reagent for introducing a linker arm between nucleotides. There are compounds that are

[0009]

[Chemical 1]

(Where n is a natural number)

[0011]

[Chemical 2]

DMTr: dimethoxytrityl group Fmoc: Fluorenylmethoxycarbonyl group

The fluorescent substance used for the labeling of the present invention may be any substance as long as it emits fluorescence, for example, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), NBD-.
Fluoride (NBD-fluoride, Sigma), Texas Red (Texas Red, Molecular Probes) and the like can be mentioned.

We have introduced a linker arm between the 5'end and the nucleotide and fluorescently labeled with FITC,
Various oligonucleotides having a plurality of fluoresceins bound thereto as shown in FIGS. 1 and 2 were prepared. That is, FIG. 1 shows eight types of fluorescent-labeled oligonucleotides (FP) prepared based on the sequence of the M4 primer represented by SEQ ID NO: 4 in the sequence listing.
1 to FP8), and FIG. 2 shows eight types of fluorescently labeled oligonucleotides (FPR1 to FPR) prepared based on the sequence of the RV primer represented by SEQ ID NO: 5 in the sequence listing.
It is a figure showing 8).

These fluorescently labeled oligonucleotides are
Fluorescence intensity, stability when forming a double chain (Tm value),
It can be evaluated by the activity of uptake by DNA polymerase when used as a primer. The present inventors evaluated these eight kinds of fluorescently labeled oligonucleotides (FP1 to FP8) shown in FIG. The results are shown in Table 1.

[0016]

[Table 1]                                 Table 1   ──────────────────────────────────     Primer Fluorescence intensity Tm (° C) Uptake efficiency                                                       (pmol / pmol)   ──────────────────────────────────       FP1 1.00 74.4 0.894       FP2 0.92 72.1 0.728       FP3 1.66 72.5 0.625       FP4 1.44 72.9 0.829       FP5 1.74 70.7 0.630       FP6 1.08 69.3 0.659       FP7 1.50 69.3 0.577       FP8 1.48 67.7 0.423   ──────────────────────────────────

As a result, the fluorescence intensity was 1 for fluorescein.
It is stronger to introduce more than one, but
Even if three or more fluoresceins were introduced, the strength did not increase any more. In addition, a large difference was observed in the strength depending on the position where fluorescein was introduced. Further, from the Tm value, the greater the number of fluorescein introduced, and the more the number of fluorescein introduced, the closer it was to the 3'-terminal side, the poorer the stability when forming a double chain. Furthermore, the uptake activity by DNA polymerase almost corresponded to the Tm value, and the priming efficiency depended on the stability of the duplex. From the above, it was determined that the introduction position and the number of the fluorescent substance are important factors, and that one introduced at the 5'end and one introduced between the nucleotides is optimal.

Further, the ability of the fluorescent-labeled oligonucleotide as a primer can be investigated in detail by using the fluorescent-labeled oligonucleotide as a primer and performing actual sequencing using a fluorescent sequencer. To quantify the signal intensity and resolution, 1
10 minutes per 00 base pairs (approximately 15 peaks are present)
It is possible to evaluate the quality of the result of the sequencing by averaging the luminances in and calculating the value obtained by subtracting the lowest luminance between them from the average value. That is,
Even if the signal intensity is high, if the separation of each peak is poor, the lowest peak becomes high and the subtraction value becomes low, so it is presumed that this value quantifies the quality of the sequencing results. The present inventors performed sequencing using the eight types of fluorescently labeled oligonucleotides (FP1 to FP8) shown in FIG. 1 as primers [(FIG. 3) and (FIG. 4)],
The above values were calculated (Fig. 5). That is, FIG. 3 and FIG.
FIG. 3 is a diagram showing the result of sequencing by a fluorescent sequencer. FIG. 3 shows 205 minutes or less, and FIG.
It is a figure showing about a part of 00 minutes or more. Also, FIG.
Is a graph in which the vertical axis represents the relative fluorescence intensity and the horizontal axis represents the number of bases.

As a result, compared to FP1 and FP2 in which one fluorescein was introduced, good results were obtained with FP3, FP4, and FP5 in which one was introduced at the 5'end and one was introduced between nucleotides, and especially FP3 and FP4. Had high sensitivity and high accuracy up to about 700 base length. However, even if the number of fluoresceins is two, two FPs introduced between nucleotides
No good results were obtained with 6. In addition, the expected sensitivity and accuracy were not obtained with FP7 and FP8 in which the number of fluoresceins was 3 or more, but the background was high and the result was bad. These results correspond well with the above-mentioned results shown in Table 1, that is, as a fluorescent-labeled primer, an oligonucleotide in which one fluorescent substance is introduced at the 5'end and one fluorescent substance between nucleotides is most suitable. I knew it was.

The base sequence can be easily determined by preparing a kit containing the fluorescently labeled primer used in the method of the present invention. The kit may contain other reagents, and the reagents contained in the kit include DNA polymerase, deoxynucleotide triphosphate (dNTP), dideoxynucleotide triphosphate (dNTP).
dNTP), a reaction buffer, and the like. Examples of the DNA polymerase included in the kit include Klenow fragment, T7 DNA polymerase, Taq DNA polymerase, BcaBEST ^™ DNA polymerase (Takara Shuzo) and the like. The form of the reagent included in the kit may be a solution or a freeze-dried product.

[0021]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto.

Example 1 (1) Synthesis of Fluorescent Labeled Oligonucleotides and Purification Oligonucleotides were synthesized using a DNA synthesizer 3
80B type (Applied Biosystems) was used, and the phosphite amidite method was used. Aminolink 2 (Applied Biosystems) is used as a reagent for introducing a linker arm at the 5 ′ end, and Amino Modifier II is used as a reagent for introducing a linker arm between nucleotides.
(Clontech) was used. The deprotected synthetic crude was evaporated to dryness under reduced pressure, and 80 μl of 1M NaHCO ₃ / Na was added.
₂ CO ₃ buffer (pH 9.5), 160 μl of sterile water,
160 μl FITC solution [FITC 5 mg 500 μl
Dissolved in a 20% (V / V) formamide solution of
Was added to dissolve well, and the mixture was allowed to stand overnight at room temperature while protected from light. After removing unreacted FITC with Sephadex G50, the target substance was collected by reverse-phase HPLC, and FP1 shown in FIG.
~ FP8 and FPR1 to FPR8 shown in Fig. 2 were produced.

(2) Confirmation of the Number of Fluorescent Substances Introduced The fluorescently labeled oligonucleotide synthesized in Example 1- (1) was treated with 20 mM sodium acetate (pH 5.3), 0.1 mM Z.
It was dissolved in 100 μl of nCl ₂ , 0.65 units of nuclease P1 was added, and the reaction was carried out at 50 ° C. for 2 hours to decompose it into mononucleotides. Next, their fluorescence intensities were measured with a spectrofluorophotometer RF-540 (Shimadzu Corporation) at an excitation wavelength of 494 nm to confirm the number of fluoresceins bound.

Example 2 (1) Measurement of fluorescence intensity 1.3 pmol of each fluorescence-labeled oligonucleotide was added to 2.7 ml of TE buffer [10 mM Tris-HCl, 1 mM ED.
TA, pH 8.0], and the fluorescence intensity was measured in the same manner as in Example 1- (2). The relative intensity of each fluorescence-labeled oligonucleotide was calculated when the fluorescence intensity of FP1 was set to 1 (Table 1).

(2) Measurement of melting temperature of double strands Oligonucleotides complementary to the sequence of each fluorescent-labeled oligonucleotide were synthesized, and 0.2 OD units of each were mixed and annealed. Absorbance at 260 nm was recorded with increasing temperature using a spectrophotometer UV2100 spectrophotometer with Tm accessory (Shimadzu). From the temperature dependence curve of this UV absorption, T
By determining the m value, the stability of each fluorescently labeled oligonucleotide as a double strand was compared (Table 1).

(3) Incorporation of dNTP by DNA polymerase 0.2 pmol of each fluorescent-labeled oligonucleotide was annealed with 0.4 pmol of M13mp19ssDNA (Takara Shuzo), and 50 μl of 25 mM TAPS-NaOH (pH).
9.3) 2 mM MgCl ₂ , 50 mM KCl, 1 mM
DTT, 0.2 mM dATP, 0.2 mM dGTP, 0.
2 mM dTTP, 0.1 mM dCTP, 0.06 μM
In a solution containing [α- ³² P] dCTP, 0.3 unit of tack polymerase was added and reacted at 72 ° C for 20 minutes. Incorporated radioactivity was measured by a liquid scintillation counter as a phosphate insoluble fraction on a DE81 filter. Synthesized DNA per pmol of template DNA
The amount was calculated (Table 1).

(4) DNA Sequencing Using 1 pmol of each fluorescently labeled oligonucleotide, M13mp18s
The dideoxy reaction was performed using 1.4 μg of sDNA (Takara Shuzo) as a template. Amplitac Sequencing Kit (for Hitachi fluorescent sequencer) (Takara Shuzo) was used for the reaction. Fluorescent DNA sequencer S for analysis of reaction products
Q3000 (Hitachi Ltd.) was used. The brightness was averaged for 10 minutes for every 100 base pairs, and the value obtained by subtracting the minimum brightness between the averages was calculated (FIGS. 3, 4, and 5).

Example 3 Construction of Kit Fluorescent labeled primer FP4 was mixed with dNTP / ddNTP mixed solution (4 types of A, G, T and C), Bucabest DNA polymerase, 10 × reaction buffer [200 mM Tris-HC.
1 (pH 8.5), 10 mM MgCl ₂ ], M13mp18ssD
NA, stop solution (95% formamide, 20
mM EDTA and 0.05% methyl violet) were combined to construct a nucleotide sequence determination kit (60 times) (Table 2).

[0029]

[Table 2]                               Table 2 ───────────────────────────────────   dNTP / ddNTP mixed solution 4 types each 120 μl   Bucabest DNA Polymerase (2U / μl) 60μl   Primer FP4 solution (1 pmol / μl) 60 μl   10 × reaction buffer 100 μl   M13mp18ssDNA (0.2 μg / μl) 20 μl   Stop solution 900 μl ───────────────────────────────────

[0030]

INDUSTRIAL APPLICABILITY The present invention provides a highly sensitive and highly accurate nucleic acid nucleotide sequence determination method using a fluorescently labeled primer having a high signal intensity, and a kit used for the method.

[Sequence list]

SEQ ID NO: 1 Array length: 24 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: GGTTTTCCCA GTCACGACGT TGTA 24 SEQ ID NO: 2 Array length: 24 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: GAGGGTTTTC CCAGTCACGA CGTT 24 SEQ ID NO: 3 Array length: 24 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: CGACGTTGTA AAACGACGGC CAGT 24 SEQ ID NO: 4 Array length: 24 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: CGCCAGGGTT TTCCCAGTCA CGAC 24 SEQ ID NO: 5 Array length: 24 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: TTTCACACAG GAAACAGCTA TGAC 24 SEQ ID NO: 6 Array length: 24 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: GAGCGGATAA CAATTTCACA CAGG 24 SEQ ID NO: 7 Array length: 24 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: TATGTTGTGT GGAATTGTGA GCGG 24 SEQ ID NO: 8 Array length: 24 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: GCGCGCAATT AACCCTCACT AAAG 24 SEQ ID NO: 9 Array length: 24 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: AACGCGTAAT ACGACTCACT ATAG 24 SEQ ID NO: 10 Array length: 24 Sequence type: Nucleic acid Number of chains: Single chain Topology: linear Sequence type: Other nucleic acid (synthetic DNA) Array: CATACGATTT AGGTGACACT ATAG 24

[Brief description of drawings]

FIG. 1 is a diagram showing fluorescently labeled oligonucleotides FP1 to FP8.

FIG. 2 Fluorescently labeled oligonucleotides FPR1 to FPR
It is a figure showing 8.

FIG. 3 is a diagram showing a part of 205 minutes or less as a result of sequencing by a fluorescent sequencer.

FIG. 4 is a diagram showing a part of 300 minutes or more as a result of sequencing by a fluorescent sequencer.

FIG. 5 is a diagram showing the relationship between relative fluorescence intensity and the number of bases.

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Ikuno Kato, Ikunoshin Kato 3-4-1, Seta, Otsu, Shiga Prefecture, Central Research Laboratory, Mina Shuzo Co., Ltd. (56) References International Publication 91/17169 (WO, A1) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12Q 1/68 C12N 15/00-15/90

Claims (3)

(57) [Claims] 1. A method for determining a nucleotide sequence of a nucleic acid by the dideoxy method, which comprises a 5'end and 4 to 12 nucleotides from the 5'end.
A method for determining a base sequence of a nucleic acid, which comprises using as a primer an oligonucleotide in which one molecule of a fluorescent substance is bound between nucleotides at positions separated by the base. 2. Nucleic acid nucleotide sequence determination by the dideoxy method.
A primer used for the 5'end and the 5'end
Nucleotide at a position 4 to 12 bases apart from the end
An oligonuc in which one molecule of fluorescent substance is bound between each
Leotide primer . 3. A kit for determining the base sequence of a nucleic acid by the method according to claim 1 , which comprises the oligonucleotide primer according to claim 2. Decision kit.